Bottom Line:
Additional labeling is also present in endosomal vacuoles, where it is often found in clathrin-coated membrane areas.This complex(es) binds exogenously added alphaSNAP and NSF and dissociates in the presence of ATP, but not ATPgammaS.These results support a role for syntaxin 13 in membrane fusion events during the recycling of plasma membrane proteins.

ABSTRACTEndocytosis-mediated recycling of plasma membrane is a critical vesicle trafficking step important in diverse biological processes. The membrane trafficking decisions and sorting events take place in a series of heterogeneous and highly dynamic organelles, the endosomes. Syntaxin 13, a recently discovered member of the syntaxin family, has been suggested to play a role in mediating endosomal trafficking. To better understand the function of syntaxin 13 we examined its intracellular distribution in nonpolarized cells. By confocal immunofluorescence and electron microscopy, syntaxin 13 is primarily found in tubular early and recycling endosomes, where it colocalizes with transferrin receptor. Additional labeling is also present in endosomal vacuoles, where it is often found in clathrin-coated membrane areas. Furthermore, anti-syntaxin 13 antibody inhibits transferrin receptor recycling in permeabilized PC12 cells. Immunoprecipitation of syntaxin 13 revealed that, in Triton X-100 extracts, syntaxin 13 is present in a complex(es) comprised of betaSNAP, VAMP 2/3, and SNAP-25. This complex(es) binds exogenously added alphaSNAP and NSF and dissociates in the presence of ATP, but not ATPgammaS. These results support a role for syntaxin 13 in membrane fusion events during the recycling of plasma membrane proteins.

Figure 4: Brefeldin A and nocodazole alter the subcellular localization of syntaxin 13. NRK cells, untreated (A and B) or treated with 5 μg/ml BFA (C and D) or nocodazole (E and F), were fixed with 4% paraformaldehyde, permeabilized with saponin, and costained for syntaxin 13 (A, C, and E) and TfR (B, D, and F). This was followed by incubation with Texas red-labeled anti–rabbit IgG and FITC-labeled anti–mouse IgG antibodies before processing for confocal microscopy. Bars, 5 μm.

Mentions:
The dynamics of membrane proteins in the presence of brefeldin A (BFA) and nocodazole can reveal features of their native localization and life cycle. BFA causes a block in ER to Golgi trafficking resulting in the redistribution of Golgi proteins back to the ER (46). At the same time in some cells it collapses the TGN and endosomes onto the microtubule-organizing center (35, 45). Indeed, treatment of NRK cells with 5 μg/ml BFA resulted in the redistribution of syntaxin 13 staining into a compact spot in the center of the cell, where it remained colocalized with the TfR (Fig. 4, compare A and B with C and D). This result shows that syntaxin 13 is not associated with Golgi but might instead reside in the TGN and/or endosomal compartments. To further investigate this issue, we treated NRK cells with the microtubule-depolymerizing agent nocodazole. The integrity of the endosomal compartment is dependent on an intact microtubule network (67) and depolymerization of microtubules causes its disintegration into multiple vesicular structures, scattered throughout the cytoplasm of the cell. Treatment of NRK cells with nocodazole converted the syntaxin 13–positive perinuclear compartment into multiple vesicular structures (Fig. 4 E), characteristic of nocodazole-induced changes of endosomal markers. Indeed, most of these vesicular structures also contain the TfR (Fig. 4 F). Thus, the above results strongly suggest that syntaxin 13 is localized to endosomes.

Figure 4: Brefeldin A and nocodazole alter the subcellular localization of syntaxin 13. NRK cells, untreated (A and B) or treated with 5 μg/ml BFA (C and D) or nocodazole (E and F), were fixed with 4% paraformaldehyde, permeabilized with saponin, and costained for syntaxin 13 (A, C, and E) and TfR (B, D, and F). This was followed by incubation with Texas red-labeled anti–rabbit IgG and FITC-labeled anti–mouse IgG antibodies before processing for confocal microscopy. Bars, 5 μm.

Mentions:
The dynamics of membrane proteins in the presence of brefeldin A (BFA) and nocodazole can reveal features of their native localization and life cycle. BFA causes a block in ER to Golgi trafficking resulting in the redistribution of Golgi proteins back to the ER (46). At the same time in some cells it collapses the TGN and endosomes onto the microtubule-organizing center (35, 45). Indeed, treatment of NRK cells with 5 μg/ml BFA resulted in the redistribution of syntaxin 13 staining into a compact spot in the center of the cell, where it remained colocalized with the TfR (Fig. 4, compare A and B with C and D). This result shows that syntaxin 13 is not associated with Golgi but might instead reside in the TGN and/or endosomal compartments. To further investigate this issue, we treated NRK cells with the microtubule-depolymerizing agent nocodazole. The integrity of the endosomal compartment is dependent on an intact microtubule network (67) and depolymerization of microtubules causes its disintegration into multiple vesicular structures, scattered throughout the cytoplasm of the cell. Treatment of NRK cells with nocodazole converted the syntaxin 13–positive perinuclear compartment into multiple vesicular structures (Fig. 4 E), characteristic of nocodazole-induced changes of endosomal markers. Indeed, most of these vesicular structures also contain the TfR (Fig. 4 F). Thus, the above results strongly suggest that syntaxin 13 is localized to endosomes.

Bottom Line:
Additional labeling is also present in endosomal vacuoles, where it is often found in clathrin-coated membrane areas.This complex(es) binds exogenously added alphaSNAP and NSF and dissociates in the presence of ATP, but not ATPgammaS.These results support a role for syntaxin 13 in membrane fusion events during the recycling of plasma membrane proteins.

ABSTRACTEndocytosis-mediated recycling of plasma membrane is a critical vesicle trafficking step important in diverse biological processes. The membrane trafficking decisions and sorting events take place in a series of heterogeneous and highly dynamic organelles, the endosomes. Syntaxin 13, a recently discovered member of the syntaxin family, has been suggested to play a role in mediating endosomal trafficking. To better understand the function of syntaxin 13 we examined its intracellular distribution in nonpolarized cells. By confocal immunofluorescence and electron microscopy, syntaxin 13 is primarily found in tubular early and recycling endosomes, where it colocalizes with transferrin receptor. Additional labeling is also present in endosomal vacuoles, where it is often found in clathrin-coated membrane areas. Furthermore, anti-syntaxin 13 antibody inhibits transferrin receptor recycling in permeabilized PC12 cells. Immunoprecipitation of syntaxin 13 revealed that, in Triton X-100 extracts, syntaxin 13 is present in a complex(es) comprised of betaSNAP, VAMP 2/3, and SNAP-25. This complex(es) binds exogenously added alphaSNAP and NSF and dissociates in the presence of ATP, but not ATPgammaS. These results support a role for syntaxin 13 in membrane fusion events during the recycling of plasma membrane proteins.